The type 1 human immunodeficiency virus (HIV-1) has been etiologically linked with the Acquired Immune Deficiency Syndrome (AIDS). HIV-l infection of permissive CD4+ cellular targets may lead either to a latent or to a lytic form of infection. The overall level of HIv-l replication is determined both by the state of host cell activation and by the concerted action of a series of HIV-l encoded regulatory proteins (tat, rev, nef). While the tat and rev proteins serve as positive factors augmenting viral gene expression and the synthesis of viral structural proteins respectively, the nef gene product appears to represent a "negative factor" that down-regulates viral replication. The principal goal of these proposed studies is to explore the function and mechanism of action of the HIv-l nef protein and to determine its possible role in the initiation or maintenance of viral latency. To approach these questions, wild type and mutant nef expression vectors will be constructed and used to prepare stable nef expressing human T and macrophage cell lines. Nef effects in primary human T lymphocytes will also be studied using a transient expression technique which permits high efficiency gene transfer in these cells. The nef protein will be specifically analyzed for effects on: (1) the overall levels of HIv-l replication, (2) mitogen induced activation of the HIv-l LTR, (3) the specific binding of cellular proteins to the HIv-l enhancer and trans-acting responsive (TAR) region and (4) the biological activity of the HIV- l tat and rev proteins. In view of its structural similarity to the recently recognized family of small guanine nucleotide binding proteins (G proteins), potential inhibitory effects of nef on the generation of the "second messengers" involved in T cell activation will also be evaluated. These studies will include the analysis of nef effects on T cell antigen receptor and CD2 mediated intracellular signalling with particular emphasis on altered calcium mobilization and phosphatidyl inositol turnover. Together, these studies should serve to provide a clearer understanding of the biological function of the nef protein, its mechanism of action, and its potential involvement in HIV-l latency.